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LMBP plasmid details

Last data update: 26 September 2020 04:22 CEST

Plasmid name: pLPPsOmpArPDI (LMBP 3056)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p3056.gb (View with Genome Compiler)
p3056.txt
p3056.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Escherichia coli outer membrane protein II gene (ompA); signal sequence
Rat protein disulfide isomerase cDNA (rPDI); mature sequence
Escherichia coli lac repressor gene (lacI)
Promoter: Escherichia coli lipoprotein promoter (lpp)
Escherichia coli lac operon promoter; mutant (lacUV5)
Escherichia coli lac repressor promoter; mutant (lacI(q))
Ribosome
binding site:
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)
Terminator: Escherichia coli lipoprotein terminator (lpp)
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Parental clone: pLPPHIs; pSE380rPDI
Further information: The plasmid was constructed as follows: 1) pSE380rPDI was cleaved with BanI, filled in with Klenow DNA polymerase and further digested with BglII; 2) subsequently, the BanI (nucleotide position 5563) - BglII fragment encoding rat protein disulfide isomerase was ligated to the SacI (blunted with T4 DNA polymerase) - BamHI vector fragment of pLPPHIs.
Expression of the fusion gene, under control of both the lpp and lacUV5 promoter, yields high levels of enzymatically active rat protein disulfide isomerase (from which the ompA signal peptide is precisely removed) in the bacterial periplasm.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727428.1.
In pLPPsOmpArPDI, the E. coli ompA signal sequence was fused in phase to the nucleotide sequence encoding the last two amino acids of the rat PDI signal sequence, followed by the rat PDI mature sequence. The nucleotide sequence at this fusion was confirmed by DNA sequence analysis.
The nucleotide sequence of the rPDI cDNA corresponds with the EMBL Nucleotide Sequence Database accession number X02918.1.
The nucleotide sequences of the 5' and 3' ends of the E. coli lacI insert, analysed by Roche Research USA, were kindly provided by Dr Yves Guisez (Roche Research Ghent). The unknown nucleotides in these analyses as well as some single nucleotides in other parts of the vector were replaced by the corresponding nucleotides of the E. coli K12 genome sequence obtained from the EMBL Nucleotide Sequence Database accession number U00096.2. However, the biological material deposited in the BCCM/GeneCorner collection seems not to correspond to the sequence analysis results of Roche Research USA (see info in the field 'Authenticity').
EMBL Accession number: X02918.1, view at EMBL, GenBank, DDBJ
U00096.1, view at EMBL, GenBank, DDBJ
LT727428.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 06/05/2008
Sequence detail:
Nucleotide sequence at the fusion between the ompA signal sequence and the rPDI mature sequence:

      --- ompA signal sequence --> -srPDI> --- rPDI mature sequence -->
5' ... TTC.GCT.ACC.GTA.GCG.CAG.GCC.GGC.GCC.GAC.GCT.CTG.GAG.GAG.GAG.GAC ... 3'
       Phe Ala Thr Val Ala Gln Ala Gly Ala Asp Ala Leu Glu Glu Glu Asp
                                 
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: AatII/KspAI, AccI, AdeI/PstI, AdeI/XbaI, Alw44I, Alw44I/KpnI, AvaI, BamHI/XbaI, BshTI, BshTI/EcoRI, CaiI/NdeI, Eco130I/Eco47III, EcoNI, HindII and KpnI/KspAI.

The plasmid seems to be approx. 500 bp larger as compared to the compiled nucleotide sequence. The extra nucleotides are located in the vector part of the plasmid between the BshTI site at position 2375 and the KpnI site at position 2504.

The restriction site analysis pattern revealed also the presence of an extra AccI, BshTI, EcoNI, HindII and KpnI site. These extra sites are located in the unknown nucleotide sequence of the approx. 500 bp fragment.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr K. De Sutter(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: De Sutter et al., Gene 141 (1994), 163-170 [PMID: 8163184]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061
Host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Related host reference: Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: -

Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.

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